GSM8021361: SpySuz12_H3K27me3_ChIP; Mus musculus; ChIP-Seq
Mouse embryonic stem cell lines (pSM33) were crosslinked in either 1% formaldehyde or 1% formaldehyde and 2 mM DSG (Thermo #2059). Briefly, cells were washed with room temperature 1X PBS and then incubated in crosslinker solution in PBS at room temperature with gentle rocking for 45 minutes. Cells were then washed with room temperature 1X PBS. After washing, a 1% formaldehyde solution was then added on top of cells and further incubated at room temperature for 10 minutes. To quench the formaldehyde crosslinking, 2 mL of a 2.5M Glycine solution was added to each plate and incubated for an additional 5 minutes. After quenching, cells were washed three times in 1X cold PBS. After the last wash, 7.5 mL of scraping buffer (1X PBS + 0.5% BSA) was added to cells. Cells were then scraped using a rubber policeman, aliquoted into 10 million cell aliquots, and flash frozen until ChIP was performed. To solubilize chromatin and fragment DNA, cells were lysed and then sonicated. To begin nuclear fractionation, cells were resuspended in 1 mL of Gagnon HLB Buffer + 1X PIC and incubated on ice for 10 minutes. Samples were briefly vortexed and centrifuged at 1250g for 3 minutes at 4°C. Samples were then resuspended in 600 μL of mammalian lysis buffer (1% TritonX-100, 0.1% sodium deoxycholate, 0.1% SDS, 150mM NaCl, and 50mM HEPES pH 7.5) + PIC and transferred to 15 mL Diagenode conical tubes. Following transfer, cells were sonicated using a Bioruptor waterbath sonicator (in 15 mL tubes with adapters) at max intensity for 30 seconds, followed by either 30 seconds of rest for 27 cycles (1% formaldehyde samples) or 30 seconds of rest for 36 cycles (1% formaldehyde + DSG samples). Samples were then transferred to 1.5 mL tubes and cleared of insoluble material by pelleting at 13000 rpm for 10 minutes at 4°C. Supernatants were mixed with 1800 μL HBSS (Thermo Scientific) + 1X PIC and 2400 μL 2X RIPA + 1X PIC. Lysate was incubated overnight with Invitrogen M-280 Sheep Anti-Rabbit IgG Dynabeads coupled with 5 μg of H3K27me3 antibody (Active Motif, 39155) or 5 μg of Goat anti-V5 antibody (Bethyl, A190-119A). Samples were washed with 1 mL of low salt buffer, high salt buffer, LiCl wash buffer, and finally TE buffers. Samples were reverse-crosslinked and Proteinase K-digested overnight at 65°C, and DNA was subsequently purified with Zymo Clean and Concentrate.  Post reverse-crosslinking ChIP libraries were constructed as previously described. Briefly, purified DNA was end repaired and dA-tailed using 1X NEBNext Ultra II End Repair/dA-Tailing Module (NEB, E7546L). DNA adaptors were then ligated to each sample and cleaned up using 0.7X SPRI (AMPure XP) followed by a repeat clean-up with 1X SPRI. PCR amplification was achieved using primers that add the indexed full Illumina adaptor sequences.